A major premise of this proposal is that RNAs present in biofluids represent a window into various cellular processes that exist in multiple tissues that release such RNAs. If we can understand how these RNAs end up outside the cell, which RNAs are present under different conditions (like how these RNAs change in the presence of certain diseases or respond to certain cellular signals like those present during immune signaling or during developmental or stem cell processes etc.) then we can provide assays for these accessible substances that give information about the whole organism's physiology. We have found that oncogenic KRAS mutations that occur in colorectal cancer can regulate miRNAs, mRNAs and long RNAs secreted from tumor cells in exosomes. In these proposals we will determine the sequence of tumor cell secreted extracellular exosomal RNAs (eRNAs-project 1) and those packaged RNAs that travel into the circulatory system (cRNAs-project 2) and how these RNAs change during cancer progression. This will be done by comprehensive RNAseq analysis. Based on this information we will find sequences associated with these RNAs that traffic them to exosomes and test the role of specific RNA processing and trafficking proteins in delivering these RNAs to exosomes, leading to their secretion out of the cell (project 2). We will pick 10 RNA-exosomal targets that will have their trafficking into the blood system modeled in vivo; testing the roles of RNA targeting sequences and RNA-associated proteins in delivering these RNAs into the blood (project 1). Finally the trafficking of these RNAs into the circulatory system and how oncogenic colorectal cancer mutations affect these RNAs and their associated trafficking mechanisms will be tested in primary human tumor xenografts (project 1). These experiments have far reaching implications for biomarker research, creates tools for analyzing and understanding the function of these secreted RNAs in cancer and in other biological paradigms for RNA secretion. We are confident that these specific RNA trafficking mechanisms will be acting in other tissues in the body and are of general relevance to multiple fields.